BACKGROUND: Despite the ability of antiretroviral therapy (ART) to inhibit HIV replication in infected patients, preventing disease progression, the virus persists in a stable reservoir and rebounds to pretherapy levels if ART is interrupted. Lifelong therapy is expensive and risks the emergence of drug resistance and toxic side effects. Consequently, identification of strategies to eliminate the reservoir is a major priority of HIV research. Recently, we and others showed that HIV-infected cells can clonally expand and persist despite ART, and that the proviral integration site may influence this phenomenon (Maldarelli et al., Science 345:179-183, 2014; Wagner et al., Science 345:570-573, 2014). In one case, an expanded provirus was shown to match the single viral variant present at detectable levels in the persistent viremia during ART; furthermore, we showed that the virus particles produced by the clonally expanded cells were replication competent (Simonetti et al., PNAS 113:1883-1888, 2016). This is one example where the source of infectious virus in blood has been traced to a clone of infected cells carrying a mostly latent provirus. A major focus of this project is to determine how commonly such clonally expanded, infected cells that persist during ART carry replication-competent proviruses and in which tissues and cell types they persist. ____Although there is considerable patient-to-patient variation, the frequency of resting CD4+ T cells that harbor HIV proviruses detectable by PCR has been very roughly estimated to average about 1 cell in 1000; however, the number of latently infected cells carrying replication-competent proviruses has been reported to be much lower (Ho et al., Cell 155:540-551, 2013). The difference is due to the presence of a large number of defective proviruses. Ya-Chi Ho and colleagues described the proviruses in resting CD4+ T cells that were not induced to produce replication-competent virus after a single round of maximal T-cell activation (Ho et al., Cell 155:540-551, 2013). Almost half of these proviruses had large internal deletions that preclude replication, while another third were lethally hypermutated by the host restriction factor APOBEC3G. Other defects and further analyses brought the fraction of defective proviruses up to 98% (Bruner et al., presentation at 2016 Conference on Retroviruses and Opportunistic Infections). Additionally, Ho et al. found that some of the intact proviruses were capable of producing infectious virions following a second round of activation (Cell 155:540-551, 2013), even though they had not been induced by the prior activation. This result leads to the question of how commonly the expanding clones described by Maldarelli et al. and Wagner et al. carry intact and replication-competent proviruses. To answer this question, we developed a novel method called the full-length integrated proviral single-genome sequencing (FLIP-SGS) assay, which allows us to analyze the proviruses in highly expanded clones like those identified by Drs. Frank Maldarelli and Stephen Hughes (HIV Dynamics and Replication Program) using the integration sites assay (ISA). The FLIP-SGS assay will determine if HIV proviruses in expanding clones that persist during ART have intact sequences or contain lethal mutations or deletions. We will further determine if, when activated, such clones are capable of producing infectious virions. If we show that latent, intact proviruses in infected cells commonly undergo clonal expansion, it will mean that strategies intended to cure patients will have to not only block viral replication, but also cope with the proliferation of these latently infected cells. ____ACCOMPLISHMENTS: We found (Kearney et al., PLoS Pathog. 10:e1004010, 2014) that the initially diverse pre-ART plasma virus population becomes more homogeneous with time, with the appearance, in many patients, of rakes of identical sequence, as originally reported by others (Bailey et al., J. Virol. 80:6441-6457, 2006). This observation suggests that there is a dynamic balance between the clonal expansion and death of infected cells during ART. It is not yet known how commonly expanded clones carry replication-competent proviruses or if they carry HIV proviruses with CTL escape mutations that may give them a survival advantage. However, to date we have identified several such cases (Simonetti et al., PNAS 113:1883-1888, 2016; Bui et al., PLoS Pathog. 13:e1006230, 2016; Patro et al., PNAS, accepted upon revision). Our current studies aim to determine if expanded clones sustain the HIV-1 reservoir in all infected individuals. ____To identify the possible sources of persistent viremia and to investigate the effect of ART on viral replication, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low (van Zyl et al., J. Clin. Invest. 127:3827-3834, 2017). Eight children started ART at 10 months of age and had plasma viremia suppressed for 7-9 years. Two children had uncontrolled viremia for 15 and 30 months, respectively, before viremia suppression. The latter two children showed clear evidence of virus evolution, whereas none of the eight children suppressed on ART showed virus evolution by multiple methods of analysis. These suggest that ongoing HIV replication in the blood is not common on ART and is, therefore, not a significant barrier to curing HIV-1 infection. ____To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during ART, we looked for evidence of viral replication in five donors after up to 13 years of viral suppression (McManus et al., J. Clin. Invest. epub before print). We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was detected in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART. ____We applied the FLIP-SGS method to lymph node and PBMCs from five ART-treated donors to determine if groups of identical sub-genomic sequences in the two compartments are the result of clonal expansion of infected cells or a genetic bottleneck on the virus (Patro et al., PNAS, accepted upon revision). We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T-cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found two clones from one donor that carried identical proviruses except for nonoverlapping deletions from which we could infer the sequence of the intact parental provirus. Thus, FLIP-SGS can be used for viral paleontology to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrated that identical sequences observed by standard SGS are not always sufficient to established proviral clonality.